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Bute to the reduction of toxic oligomers in the cytoplasm of Bute to the reduction of toxic oligomers in the cytoplasm of mammalian cells; the expression of Hsp27 or of the yeast Hsp104 can reduce toxicity associated with -Syn oligomers in human H4 neuroglioma cells or rat models of PD, respectively (48, 49). However, the contribution of sHSPs is expected to be limited to the passive prevention of protein aggregation as, unlike Hsp70/Hsp40, they cannot use the energy of ATP hydrolysis to unfold misfolded proteins. It can, however, stabilize and deliver misfolded proteins to the Hsp70/Hsp40 chaperone system (50). True functional homologues of ClpB acting together with Hsp70/Hsp40/NEF in the disaggregation of large compact aggregates (51) have been identified only in bacteria in the organelles and the cytoplasm of plants, yeast, and fungi (for review, see Ref. 5). In animal cells, among the many cytoplasmic AAA proteins with conserved Walker motives, none has been convincingly shown thus far to function as true ClpB/Hsp104 homologues. Our immunoblot analysis showed that, unlike with a classic heat-aggregated chaperone substrate, -Syn oligomers remained resistant to the unfolding action of ClpB, which remained unable to improve the limited disaggregating activity of DnaK/DnaJ/GrpE alone (data not shown). Hence, the Hsp70/40/NEF remains thus the only known chaperone machinery in the cytoplasm of mammalian cells that can use theVOLUME 285 ?NUMBER 49 ?DECEMBER 3,38180 JOURNAL OF BIOLOGICAL CHEMISTRY-Synuclein Oligomers Inhibit Chaperone Activity of Hsp70/energy of ATP hydrolysis to unfold toxic misfolded protein conformers, such as -Syn oligomers, into non-toxic natively refoldable or protease-degradable species (5, 27). Recruiting Hsp70/Hsp40/NEF to Reduce PD Is a https://britishrestaurantawards.org/members/singer20europe/activity/356443/ Promising Therapeutic Approach--An age-dependent progressive failure of the protein clearance machinery, including the Hsp70/ Hsp40 chaperone system, has been shown in aging organisms (for review, see Ref. 52). Interestingly, in agreement with our finding that J-domain co-chaperones play a central role in aggregate recognition and processing, a systematic screen in human cell cultures expressing disease-associated polyglutamine proteins (polyQ) revealed that two human DnaJ homologues, DNAJB6b and DNAJB8, are effective suppressors of polyQ aggregation and toxicity (53). This suggests new avenues of therapeutic approaches using chaperone-inducing drugs, such as Hsp90 inhibitors (54) or non-steroidal anti-inflammatory drugs (55). Alternatively, vectors could mediate the specific expression of particular effective J-domain co-chaperones in aging or diseased neurons, thus, targeting more effectively the unfoldase activity of the cytoplasmic Hsp70/Hsp40s to the cytotoxic -Syn oligomers in PD. Mechanistic Implications; J-domain Co-chaperones May Preferably Bind Misfolded Structures--Although the native unstructured monomeric form of human -Syn apparently optimally exposes a typical high affinity binding motive, 32 KTKEGVLYVGSKTR45, for the potential binding and locking of Hsp70 (29) (see supplemental Fig. S6), we found that even in large molar excess, it did not inhibit the ATP- and DnaK/ DnaJ/GrpE-mediated unfolding/refolding reaction of classic amenable chaperone substrates. This is consistent with NMR and gel filtration studies that show no interaction of Hsp70 with monomeric -Syn (56, 57). Our work further shows that DnaJ too has no apparent affinity for the unstructured monomeric form of -Syn. In contrast, we found that the three J-domain co-c.